1. Field of the Invention
The invention is directed to purified and isolated novel members of the metalloproteinase-disintegrin family, specifically, SVPH3-13 and SVPH3-17 polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.
2. Description of Related Art
Proteins containing disintegrin and metalloproteinase domains
Metalloproteinases are a group of proteinases characterized by the presence of a metal prosthetic group. Despite this basic similarity, the group, which includes proteinases from snake venom, numerous microbial proteinases, and vertebrate and bacterial collagenases, would seem to present proteinases of seemingly widely varying activities. For example, snake venom proteases are metalloproteinases that affect cell-matrix interactions. Snake venom also includes xe2x80x9cdisintegrins,xe2x80x9d a class of low molecular weight, Arg-Gly-Asp (RGD)-containing, cysteine-rich peptides which bind to integrins (a family of molecules involved in cell-to-cell adhesion, cell-to-matrix adhesion, and inflammatory responses) expressed on cells.
Also included are the membrane-anchored ADAMs (A Disintegrin And Metalloproteinase), which are multimeric molecules consisting of metalloproteinase, disintegrin-like, cysteine rich, and epidermal growth factor domains. See Black, R. A. and White, J. M., (1998) xe2x80x9cADAMs: focus on the protease domain,xe2x80x9d Curr Opin Cell Biol 10, 654-659 (in process); Wolfsberg, T. G. and White, J. M. (1996) xe2x80x9cADAMs in fertilization and development,xe2x80x9d Dev Bio 180, 389-401, all of which are herein incorporated by reference. The ADAMs family includes fertilin-xcex1 and meltrin-xcex1, both of which are involved in membrane or cell-cell fusion. Specifically, the disintegrin domain of fertilin-xcex1 and meltrin-xcex1 have been implicated in sperm/egg fusion and myoblast fusion, respectively. implicated in sperm/egg fusion and myoblast fusion, respectively.
Another member of this family, ADAM 10/KUZ, has been identified as being involved in neurogenesis. (Id.)
The ADAMs family of metalloproteinase-disintegrins also share homology with the snake venom protease family (SVPH). In some snake venom protease members, the disintegrin domain prevents platelet aggregation and thus acts as an anti-coagulant. ADAMs and SVMPs share an extended catalytic site sequence and an activation mechanism, which involves proteolytic removal of the Pro domain. (Id.) In vitro cleavage of extracellular matrix molecules and cell surface proteins by ADAMs and SVMPs has been seen.
The ADAMs family members display a common domain organization, corresponding to potential proteolysis, adhesion, signaling, and fusion functions of these proteins. (Id.) Since several ADAMs have been shown to interact with integrins, bidirectional signaling is possible. (Id.) Some of the ADAMs are active proteinases.
Roles for ADAMs in matrix degradation, cell migration, and localized shedding of proteins including cytokines and growth factors have been reported. (Id.) For example, tumor necrosis factor xcex1 is cleaved by ADAM 17 to release a soluble form of the protein (Black et al., Nature 385:729-733, 1997; and Moss et al., Nature 385:733-736, 1997).
ADAMS 1-6 have been implicated in fertilization and/or spermatogenesis (Barker, H. L., Perry, A. C., Jones, R., and Hall, L., Biochim Biophys Acta, 1218, 429-31, 1994; Blobel, C. P., Wolfsberg, T. G., Turck, C. W., Myles, D. G., Primakoff, P., and White, J. M., Nature, 356, 248-252, 1992; Evans, J. P., Schultz, R. M., and Kopf, G. S., J. Cell Sci, 108, 3267-3278, 1995; Perry, A. C., Barker, H. L., Jones, R., and Hall, L., Biochim Biophsy Acta, 1207, 134-137, 1994; Perry, A. C., Gichuhi, P. M., Jones, R., and Hall, L., Biochem J., 307, 843-850, 1995; Perry, A. C., Jones, R., and Hall, L., Biochem J., 312, 239-244, 1995; Wolfsberg, T. G., Bazan, J. F., Blobel, C. P., Mules, D. G., Primakoff, P., and White, J. M., Proc Natl Acad Sci USA, 90, 10783-10787, 1993; and Wolfsberg, T. G., Straight, P. D., Gerena, R. L., Huovila, A. P., Primakoff, P., Myles, D. G., and White, J. M., Dev Biol, 169, 378-383, 1995).
The ADAMs family also includes the TNF-xcex1 converting enzymes (TACE). See Blobel, C. P., (1997), xe2x80x9cThe Metallo-disintegrins: Links to cell adhesion and cleavage of TNFa and notch,xe2x80x9d Cell 90,589-592. TACE is required for the shedding of membrane proteins including TNF xcex1, p80 TNFR, p60TNFR, L-selectin, type II IL-IR, and xcex2-amyloid precursor protein.
Given the significant function of metalloproteinases in membrane and cell-cell fusion, cellular adhesion, shedding of membrane proteins, anti-coagulation, and neurogenesis there is a need in the art for additional metalloproteinases, of the ADAMs family and/or of the SVPH family members, including the discovery, identification, and roles of new proteins within these families.
Molecular weight and isoelectric point determinations
In another aspect, the identification of the primary structure, or sequence, of an unknown protein is the culmination of an arduous process of experimentation. In order to identify an unknown protein, the investigator can rely upon a comparison of the known protein to known peptides using a variety of techniques known to those skilled in the art. For instance, proteins are routinely analyzed using techniques such as electrophoresis, sedimentation, chromatography, sequencing and mass spectrometry.
In particular, comparison of an unknown protein to polypeptides of known molecular weight allows a determination of the apparent molecular weight of the unknown protein (T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)). Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein (New England Biolabs Inc. Catalog:130-131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758). However, the molecular weight standards may not correspond closely enough in size to the unknown protein to allow an accurate estimation of apparent molecular weight. The difficulty in estimation of molecular weight is compounded in the case of proteins that are subjected to fragmentation by chemical or enzymatic means, modified by post-translational modification or processing, and/or associated with other proteins in non-covalent complexes.
In addition, the unique nature of the composition of a protein with regard to its specific amino acid constituents results in unique positioning of cleavage sites within the protein. Specific fragmentation of a protein by chemical or enzymatic cleavage results in a unique xe2x80x9cpeptide fingerprintxe2x80x9d (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977; M. Brown et al., J. Gen. Virol. 50:309-316, 1980). Consequently, cleavage at specific sites results in reproducible fragmentation of a given protein into peptides of precise molecular weights. Furthermore, these peptides possess unique charge characteristics that determine the isoelectric pH of the peptide. These unique characteristics can be exploited using a variety of electrophoretic and other techniques (T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)).
Fragmentation of proteins is further employed for amino acid composition analysis and protein sequencing (P. Matsudiara, J. Biol. Chem. 262:10035-10038, 1987; C. Eckerskom et al., Electrophoresis 1988, 9:830-838, 1988), particularly the production of fragments from proteins with a xe2x80x9cblockedxe2x80x9d N-terminus. In addition, fragmented proteins can be used for immunization, for affinity selection (R. A. Brown, U.S. Pat. No. 5,151,412), for determination of modification sites (e.g. phosphorylation), for generation of active biological compounds (T. D. Brock and M. T. Madigan, Biology of Microorganisms 300-301 (Prentice Hall, 6d ed. 1991)), and for differentiation of homologous proteins (M. Brown et al., J. Gen. Virol. 50:309-316, 1980).
In addition, when a peptide fingerprint of an unknown protein is obtained, it can be compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D. Fenyo et al., Electrophoresis 19:998-1005, 1998). A variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (prospector.uscf.edu), Multildent (expasy.ch/sprot/multiident), PeptideSearch (mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form), and ProFound (chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance. The programs compare these molecular weights to protein molecular weight information stored in databases to assist in determining the identity of the unknown protein. Accurate information concerning the number of fragmented peptides and the precise molecular weight of those peptides is required for accurate identification. Therefore, increasing the accuracy in determining the number of fragmented peptides and their molecular weight should result in enhanced likelihood of success in the identification of unknown proteins.
In addition, peptide digests of unknown proteins can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec. 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994); J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec. 11:1067-1075 (1997)). Searching programs that can be used in this process exist on the Internet, such as Lutefisk 97 (Isbc.com:70/Lutefisk97), and the Protein Prospector, Peptide Search and ProFound programs described above. Therefore, adding the sequence of a gene and its predicted protein sequence and peptide fragments to a sequence database can aid in the identification of unknown proteins using tandem mass spectrometry.
Thus, there also exists a need in the art for polypeptides suitable for use in peptide fragmentation studies, for use in molecular weight measurements, and for use in protein sequencing using tandem mass spectrometry.
The invention aids in fulfilling these various needs in the art by providing new members of the metalloproteinase-disintegrin family, specifically, SVPH3-13 and SVPH3-17 nucleic acids and polypeptides encoded by these nucleic acids. Particular embodiments of the invention are directed to an isolated SVPH3-13 or SVPH3-17 nucleic acid molecule comprising the DNA sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:5 and an isolated SVPH3-13 or SVPH3-17 nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6, as well as nucleic acid molecules complementary to these sequences. The invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells stably or transiently transformed or transfected with these vectors.
In addition, the invention encompasses methods of using the nucleic acids noted above to identify nucleic acids encoding proteins having proteinase activity; to identify human chromosome number 2 or 7; to map genes on human chromosome number 2 or 7, to identify genes associated with certain diseases, syndromes, or other human conditions associated with human chromosome number 2 or 7.
The invention also encompasses the use of sense or antisense oligonucleotides from the nucleic acid of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:5 to inhibit the expression of the polynucleotide encoded by the SVPH3-13 or SVPH3-17 gene.
The invention also encompasses isolated polypeptides and fragments thereof encoded by these nucleic acid molecules including soluble polypeptide portions of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6. The invention further encompasses methods for the production of these polypeptides, including culturing a host cell under conditions promoting expression and recovering the polypeptide from the culture medium. Especially, the expression of these polypeptides in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
In general, the polypeptides of the invention can be used to study cellular processes such as immune regulation, cell proliferation, cell death, cell migration, cell-to-cell interaction, and inflammatory responses.
In addition, the invention includes assays utilizing these polypeptides to screen for potential inhibitors of activity associated with polypeptide counter-structure molecules, and methods of using these polypeptides as therapeutic agents for the treatment of diseases mediated by SVPH polypeptide counter-structure molecules. Further, methods of using these polypeptides in the design of inhibitors thereof are also an aspect of the invention.
The invention further provides a method for using these polypeptides as molecular weight markers that allow the estimation of the molecular weight of a protein or a fragmented protein, as well as a method for the visualization of the molecular weight markers of the invention thereof using electrophoresis. The invention further encompasses methods for using the polypeptides of the invention as markers for determining the isoelectric point of an unknown protein, as well as controls for establishing the extent of fragmentation of a protein.
Further encompassed by this invention are kits to aid in these determinations. The invention further encompasses a kit for the determination of the molecular weights of peptide fragments of a sample protein comprising the following: a vessel; an SVPH3-13 or SVPH3-17 polypeptide; at least one enzyme selected from the group consisting of Asparaginylendoipeptidase, Arginylendopeptidase, Achrombobacter protease I, Trypsin, Staphlococcus aureus V8 protease, Endoproteinase Asp-N, and Endoproteinase Lys-C; a mutated polypeptide derived from said polypeptide by in vitro mutagenesis, wherein a site of enzymatic cleavage by the selected enzyme has been removed; and fragmented peptides derived from said peptide by enzymatic cleavage with the selected enzyme wherein said polypeptide and said sample protein are contacted with the selected protease; and wherein the protein, polypeptides, and fragmented peptides are visualized by application of the protein, polypeptides, and fragmented peptides to an acrylamide gel, resolution of the protein, polypeptides, and fragmented peptides using an electrical current, and application to the gel of a detection reagent, which stains the protein, polypeptides, and fragmented peptides.
Further encompassed by this invention is the use of the SVPH3-13 and SVPH3-17 nucleic acid sequences, predicted amino acid sequences of the polypeptide or fragments thereof, or a combination of the predicted amino acid sequences of the polypeptide and fragments thereof for use in searching an electronic database to aid in the identification of sample nucleic acids and/or proteins.
Isolated polyclonal or monoclonal antibodies that bind to these polypeptides are also encompassed by the invention, in addition the use of these antibodies to aid in purifying SVPH3-13 and SVPH3-17 polypeptides.